Tryptophan as an Auxin Precursor in Cucumber Seedlings

The conversion of tryptophan-(14)C to indoleacetic acid-(14)C in cucumber hypocotyls occurred under both sterile and non-sterile conditions. This conversion was not reduced under sterile conditions. The growth response of cucumber hypocotyl segments to exogenously supplied tryptophan was almost as great under sterile conditions as when contaminating micro-organisms were present. These data are consistent with the hypothesis that tryptophan is a normal precursor of indoleacetic acid in cucumber tissues.The conversions of tryptamine-(14)C and indoleethanol-(14)C to indoleacetic acid-(14)C also occurred under both sterile and non-sterile conditions. Indoleethanol-(14)C was formed from tryptamine-(14)C. Hypocotyl segment growth responses to tryptamine and to indoleethanol were not decreased under sterile conditions.     

Effect of Light on Mevalonic Acid Incorporation into the Phytosterols of Nicotiana tabacum L. Seedlings

Mevalonic acid-2-¹⁴C was readily incorporated into the free, esterified, and glycosidic sterol fractions of tobacco (Nicotiana tabacum L. var. Burley 21) seedlings. The time course of mevalonic acid-2-¹⁴C incorporation was different for the various individual sterols. Campesterol and sitosterol (group I) became radioactive as the free sterol and subsequently as the steryl ester. The reverse order was observed for cholesterol and stigmasterol (group II). Light stimulated the incorporation of mevalonic acid-2-¹⁴C into the group I free sterols and during the first 6 to 9 hours into the steryl esters of group II. The increase in specific radioactivity of the group II steryl esters was followed by a decline. Based on time course studies it is suggested that the group II steryl esters turn over rapidly and that light influences the rate of turnover.     

Difference between light- and wound-induced biosynthesis of α-solanine and α-chaconine in potato tubers

Potato tuber tissues can incorporate mevalonic acid-2-14C into glycoalkaloids, namely α-chaconine and α-solanine. The percent incorporation of this labeled precursor into α-chaconine in light exposed tubers is more than that of mechanically injured tubers.     

Biosynthesis of sterols and triterpenes in Pelargonium hortorum

Although all parts of the geranium plant (Pelargonium hortorum) are capable of synthesizing sterols and triterpenes and their esters in vitro from mevalonic acid-[2-¹⁴C], the aerial portions are more active than other tissues. All plant parts were shown to incorporate mevalonic acid-[2-¹⁴C] into isoprenoids for at least 3 days. The leaves and petioles had the greatest incorporation on a wet weight basis. Chopped preparations showed comparable incorporations of mevalonate whereby rootlets incorporated about one half as much as most parts; the flower petals incorporated five times the average amount. In leaves the principal sterol synthesized was sitosterol. Metabolic studies with isolated leaves indicated a fairly rapid conversion of free tetracyclic triterpenes to 4-desmethyl-sterols, while β-amyrin was synthesized at a different rate than α-amyrin. Esterified tetracyclic triterpenes exhibited only a slight amount of conversion to 4-desmethylsterols.     

Stimulation of liver cholesterol synthesis by actinomycin D

1. An eightfold increase in the incorporation of [2-(14)C]acetate into liver cholesterol in vivo was observed 24hr. after starved rats had been given actinomycin D (0.5mg./kg. of body wt.). Liver cholesterol radioactivity declined faster in the treated animals, suggesting a greater rate of cholesterol turnover. 2. Liver slices from treated animals showed a tenfold increase in the incorporation of [2-(14)C]acetate into cholesterol; conversion into CO(2) and into fatty acids was less markedly increased, and conversion into ketone bodies was not significantly affected. 3. The patterns of conversion into liver cholesterol in vivo of the lactone and the sodium salt of mevalonic acid differed markedly. The former was converted at a faster rate and to a greater extent than the latter. Treatment with actinomycin D increased the conversion of both forms of mevalonic acid into liver cholesterol, but only to a small extent. 4. Stimulation of the incorporation of acetate into cholesterol occurred at 4hr. after the administration of actinomycin D but not at 2hr. The response was abolished by the simultaneous administration of dl-ethionine or puromycin. 5. Pre-feeding with a cholesterol-rich diet greatly diminished the stimulation of conversion of acetate into cholesterol caused by actinomycin D, though it did not completely suppress it. Adrenalectomized animals responded to the drug, but much less markedly. 6. It is concluded that actinomycin D stimulates the synthesis of cholesterol in the liver at a stage in the pathway before mevalonic acid, by a mechanism that probably requires protein synthesis. A likely site would be the beta-hydroxy-beta-methylglutaryl-CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis. Some possible mechanisms by which the drug may lead to increased activity of this enzyme are considered.     

The regulation of ubiquinone synthesis in fibroblasts: The effect of modulators of β-hydroxy-β-methylglutaryl-coenzyme A reductase activity

The effect of inhibitors of β-hydroxy-β-methylglutaryl-coenzyme A (HMG-CoA) reductase such as low-density lipoprotein (LDL) and compactin were tested for their effects on the biosynthesis of ubiquinone in fibroblasts using [2-¹⁴C]acetic acid as a labeled precursor. LDL added to fibroblasts incubated in lipoprotein-deficient serum inhibited acetate incorporation into ubiquinone by 35%. Compactin, 2.5 μm, inhibited acetate incorporation by 60%. Further increases in compactin concentration up to 20 μm gradually increased the extent of inhibition but leveled off between 70 and 80%. The incorporation of ³H]mevalonic acid and 4-[U-¹⁴C]hydroxybenzoic acid into ubiquinone were determined with a range of compactin concentrations. Whereas the incorporation of [³H]mevalonate showed an apparent increase in response to compactin, the incorporation of 4-[U-¹⁴C]hydroxybenzoate into ubiquinone decreased. Both curves leveled off at concentrations of 5 μm did not significantly change with further increases in compactin concentration approaching 20 μm. Thus, the inhibition of acetate and 4-hydroxybenzoate incorporation into ubiquinone by compactin showed similar patterns. Cells incubated in lipoprotein-deficient serum compared to whole human serum showed inhibition of acetate incorporation similar to that observed previously for 4-hydroxybenzoate (9), thereby suggesting the presence of a stimulatory factor for ubiquinone biosynthesis in whole human serum. These data confirm and extend our earlier conclusions that inhibition of HMG-CoA reductase greatly affects ubiquinone synthesis in fibroblasts.     

Sterol-like compound from sensory ganglia. Effect of a nerve growth factor and insulin on its biosynthesis

1. Sensory ganglia from 8-day-old chick embryos were incubated with a specific nerve growth factor and with insulin. 2. From the total lipid extract of the ganglia a compound with steroid characteristics was isolated. 3. The synthesis of this compound, measured spectrophotometrically, diminished after addition of the nerve growth factor and insulin to the incubation medium. 4. The incorporation of sodium [2-(14)C]acetate and dl-[2-(14)C]mevalonic acid into total lipids of the sensory ganglia was stimulated by the nerve growth factor and insulin, but the radioactivity of the sterol-like compound was slightly lower. The incorporation of labelled mevalonic acid either into total lipids or into the sterol-like compound was about 25% lower. 5. About 20% of the acetate incorporated into total lipids and about 87% of the mevalonic acid were recovered in the sterol-like compound.     

The biosynthetic incorporation of the intact leucine skeleton into sterol by the trypanosomatid Leishmania mexicana

The amino acid leucine is efficiently used by the trypanosomatid Leishmania mexicana for sterol biosynthesis. The incubation of [2-(13)C]leucine with L. mexicana promastigotes in the presence of ketoconazole gave 14alpha-methylergosta-8,24(24(1))-3beta-ol as the major sterol, which was shown by mass spectrometry to contain up to six atoms of (13)C per molecule. (13)C NMR analysis of the 14alpha-methylergosta-8,24(24(1))-3beta-ol revealed that it was labeled in only six positions: C-2, C-6, C-11, C-12, C-16, and C-23. This established that the leucine skeleton is incorporated intact into the isoprenoid pathway leading to sterol; it is not converted first to acetyl-CoA, as in animals and plants, with utilization of the acetyl-CoA to regenerate 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). An inhibitor of HMG-CoA synthase (L-659,699) blocked the incorporation of [1-(14)C]acetate into sterol but had no inhibitory effect on [U-(14)C]leucine incorporation. The HMG-CoA reductase inhibitor lovastatin inhibited promastigote growth and [U-(14)C]leucine incorporation into sterol. The addition of unlabeled mevalonic acid (MVA) overcame the lovastatin inhibition of growth and also diluted the incorporation of [1-(14)C]leucine into sterol. These results are compatible with two routes by which the leucine skeleton may enter intact into the isoprenoid pathway. The catabolism of leucine could generate HMG-CoA that is then directly reduced to MVA for incorporation into sterol. Alternatively, a compound produced as an intermediate in leucine breakdown to HMG-CoA (e.g. dimethylcrotonyl-CoA) could be directly reduced to produce an isoprene alcohol followed by phosphorylation to enter the isoprenoid pathway post-MVA.     

Biosynthesis of cholesterol in rabbit reticulocytes and its exchange with plasma

When rabbit reticulocytes were incubated in normal blood plasma containing mevalonic acid-2-(14)C, radioactivity was incorporated into cholesterol, cholesteryl esters, and squalene in the cells. The squalene reached a steady level of radioactivity much more rapidly than did cholesterol. Rabbit reticulocytes which were labeled as a result of previous incubation with mevalonic acid-2-(14)C were incubated with normal autologous blood plasma. The specific activity of the cholesterol in the plasma rapidly became higher than that of the cells. This suggests that there is compartmentation of cholesterol in the reticulocyte and that a pool involved in exchange with plasma cholesterol has a specific activity which is much higher than the average for the whole cell.     

Formation of mevalonic acid, sterols and bile acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in the liver of rabbits with experimental hypercholesterolemia

The effect of cholesterol diet on the rate of mevalonic acid biosynthesis from 1-14C acetyl-CoA, 2-14C malonyl-CoA and the incorporation of these substrates into sterols and bile acids in rabbit liver were studied. Simultaneously, the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and the biosynthesis of fatty acids from acetyl-CoA and malonyl-CoA were measured. Hypercholesterolemia was found to be concomitant with the inhibition of acetyl-CoA carboxylase activity only in cell-free (700 g) and mitochondrial fractions and slightly decreased the incorporation of acetyl-CoA and malonyl-CoA into fatty acids in the postmitochondrial fraction. The HMG-CoA reductase activity in all subcellular fractions except for the postmicrosomal one was inhibited under these conditions. A significant decrease of acetyl-CoA incorporation and an increase in malonyl-CoA incorporation into mevalonic acid in all liver fractions except for microsomal one were observed in rabbits with hypercholesterolemia. These data provide evidence for the existence of two pathways of mevalonic acid synthesis from the above-said substrates that are differently sensitive to cholesterol. Cholesterol feeding resulted in a decreased synthesis of the total unsaponified fraction including cholesterol from acetyl-CoA, malonyl-CoA and mevalonic acid. The rate of incorporation of these substrates into lanosterol was unchanged. All the indicated substrates (acetyl-CoA, malonyl-CoA, mevalonic acid) are precursors of bile acid synthesis in rabbit liver. Cholesterol feeding and the subsequent development of hypercholesterolemia resulted in bile acid synthesis stimulation, preferentially in the formation of the cholic + deoxycholic acids from these precursors.     

Lysine Biosynthesis in Barley (Hordeum vulgare L.)

Lysine biosynthesis in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following precursors into the endosperm of the seedlings: acetate-1-¹⁴C; acetate-2-¹⁴C; pyruvate-1-¹⁴C; pyruvate-2-¹⁴C; pyruvate-3-¹⁴C; alanine-1-¹⁴C; aspartic acid-1-¹⁴C; aspartic acid-2-¹⁴C; aspartic acid-3-¹⁴C; aspartic acid-4-¹⁴C; α-aminoadipic acid-1-¹⁴C; and α, ε-diaminopimelic acid-1-(7)-¹⁴C. The distribution of activity in the individual carbon atoms of lysine in the different biosynthetic experiments was determined by chemical degradation. The incorporation percentages and labeling patterns obtained are in agreement with the occurrence of the diaminopimelic acid pathway. The results do not fit the incorporation percentages and labeling patterns expected if the α-aminoadipic acid pathway was operating. However, the results show that barley seedlings are able to convert a small part of the α-aminoadipic acid administered directly to lysine.     

Incorporation of dl-[2-14C]mevalonic acid lactone into β-carotene and the phytol side chain of chlorophyll in cotyledons of four species of pine seedlings

1. The incorporation of dl-[2-(14)C]mevalonic acid lactone into beta-carotene and the phytol side chain of chlorophyll has been investigated in cotyledons of four species of pine seedlings (Pinus silvestris, P. contorta, P. radiata and P. jeffrei) grown in darkness and in light. 2. The relative incorporation of label into beta-carotene and the phytol side chain of chlorophyll is similar to that observed in experiments on monocotyledons and dicotyledons. 3. The relative incorporation of (14)CO(2) into beta-carotene and phytol is much higher than the incorporation of [2-(14)C]mevalonic acid.     

Incorporation of [14C]carbon dioxide and [2-14C]mevalonic acid into terpenoids of higher plants during chloroplast development

1. The incorporation of (14)CO(2) and dl-[2-(14)C]mevalonic acid into various terpenoids in developing chloroplasts in a number of seedlings has been studied. 2. beta-Carotene and phytol (from chlorophyll) tend to be heavily labelled from (14)CO(2), whereas sterols and beta-amyrin are only slightly labelled; with dl-[2-(14)C]mevalonic acid the situation is reversed. 3. The incorporation of (14)CO(2) into terpenoids is dependent on the stage of chloroplast development, whereas that of mevalonic acid is independent of chloroplast development. 4. The uptake of (14)CO(2) into beta-carotene and phytol in mature chloroplasts is very low in monocotyledons but somewhat greater in dicotyledons. 5. The results are discussed in relation to the view that terpenoid biosynthesis in developing chloroplasts is regulated by a combination of enzyme segregation and specific membrane permeability.     

Biosynthesis of the farnesyl moiety of heme a from exogenous mevalonic acid by cultured chick liver cells

Chick embryo liver cells, when cultured for 41 h in the presence of [2-14C]mevalonic acid, took up label and incorporated radioactivity into heme a, but not into protoheme. Incubation of cells with delta-[4-14C]aminolevulinic acid (ALA) resulted in uptake of label and incorporation of radioactivity into both protoheme and heme a. These results show that both protoheme and heme a are synthesized during the incubation period, and that mevalonic acid is a specific precursor of the farnesyl moiety of heme a. Incubation of cells with [1,2-14C]acetate plus N-methyl mesoporphyrin IX, an inhibitor of heme synthesis, resulted in negligible incorporation of label into protoheme and heme a, although cellular lipids were highly labeled. This result indicates that the heme purification methods employed were capable of separating hemes from lipids, and that the measured incorporation of label into hemes from [14C]mevalonic acid and [14C]ALA was not due to lipid contamination.     

Comparative Effects of Ancymidol and Its Analogs on Growth of Peas and Ent-Kaurene Oxidation in Cell-Free Extracts of Immature Marah macrocarpus Endosperm

The plant growth retardant alpha-cyclopropyl-alpha-(4-methyoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) and a series of analogs of this substance in which one or more of the substituents were varied were tested for their comparative biological activity. The compounds were tested as inhibitors of internode elongation in peas and as inhibitors of the oxidation of ent-kaurene catalyzed by microsomal preparations from the liquid endosperm of Marah macrocarpus seeds. The relative effectiveness of a substance was generally the same as an inhibitor of the two processes. Ancymidol was the most effective. Substitution of the alcohol group of ancymidol by either methoxy or hydrogen groups reduced the activity only slightly. Substitution of the cyclopropyl group by an isopropyl moiety also had little effect on the activity. However, substitution of the cyclopropyl group with a phenyl or other aryl substituent greatly reduced the effectiveness of the analog as an inhibitor. Replacement of the 4-methoxyphenyl substituent with a similar substituent such as 4-chlorophenyl had little effect on activity, but replacement with a 2-methoxyphenyl group greatly reduced activity. Analogs in which the pyrimidyl moiety of ancymidol was modified were inactive in whole plants, but moderately active in the cell-free ent-kaurene oxidation system. The application of gibberellic acid can overcome the growth inhibitions due to treatment of the test plants with 10(-5)m or lower concentrations of the inhibitors. However, the inhibitory effects of 10(-4)m or higher concentrations of inhibitors on test plants were not overcome by the applications of exogenous gibberellic acid. These results support the idea that the effects of low concentrations of these substances on plant growth are primarily a consequence of their ability to inhibit ent-kaurene oxidation and gibberellin biosynthesis. Other modes of inhibition may operate at higher inhibitor concentrations.     

Lysine Catabolism in Barley (Hordeum vulgare L.)

Lysine catabolism in seedlings of barley (Hordeum vulgare L. var. Emir) was studied by direct injection of the following tracers into the endosperm of the seedlings: aspartic acid-3-(14)C, 2-aminoadipic acid-1-(14)C, saccharopine-(14)C, 2,6-diaminopimelic acid-1-(7)-(14)C, and lysine-1-(14)C. Labeled saccharopine was formed only after the administration of either labeled 2,6-diaminopimelic acid or labeled lysine to the seedlings. The metabolic fate of the other tracers administered also supported a catabolic lysine pathway via saccharopine, and apparently proceeding by a reversal of some of the biosynthetic steps of the 2-aminoadipic acid pathway known from lysine biosynthesis in most fungi. Pipecolic acid seems not to be on the main pathway of l-lysine catabolism in barley seedlings.     

Biosynthesis of sitosterol in barley seedlings

A method is described for the chemical synthesis of stigmasta-5,24-dien-3β-ol-[26-¹⁴C] and (24S)-24-ethylcholesta-5,25-dien-3β-ol-[26-¹⁴C] (clerosterol). 28-Isofucosterol-[7-³H₂] fed to developing barley seedlings (Hordeum vulgare) was incorporated into sitosterol and stigmasterol confirming the utilisation of a 24-ethylidene sterol intermediate in 24α-ethyl sterol production in this plant. Also, the use of mevalonic acid-[2-¹⁴C(4R)-4-³H₁] verified the loss of the C-25 hydrogen of 28-isofucosterol during its conversion into sitosterol and stigmasterol in agreement with the previously postulated isomerisation of the 24-ethylidene sterol to a Δ²⁴⁽²⁵⁾-sterol prior to reduction. However, feeding stigmasta-5,24-dien-3β-ol [26-¹⁴C] to barley seedlings gave very low incorporation into sitosterol. Attempts to trap radioactivity from mevalonic-[2-¹⁴C(4R)-4-³H₁] in stigmasta-5,24-dien-3β-ol when this unlabelled sterol was administered to barley seedlings gave only a very small incorporation although both 28-isofucosterol and sitosterol were labelled.     

Effect of halofenate and clofibrate on growth and lipid synthesis in Saccharomyces cerevisiae

Halofenate-free acid (HFA) inhibited the growth of Saccharomyces cerevisiae by 50% at a concentration of 0.34 mm. This inhibitory effect was prevented by addition of either oleate or acetate, but not by pyruvate. When cell growth was supported by oleate, HFA inhibited the incorporation of radioactive carbon from glucose-U-(14)C or pyruvate-2-(14)C into fatty acids and sterols. The incorporation of radioactive carbon into fatty acids and sterols from acetate-2-(14)C was unaffected by the compound. When cell growth was supported by either oleate or acetate, HFA inhibited the conversion of pyruvate-1-(14)C to (14)CO(2). These results suggest that HFA inhibits the conversion of pyruvate to acetate in yeast. Partially purified yeast pyruvate dehydrogenase was inhibited 50% by 5.5 mm HFA; however, the concentration required for 50% inhibition was considerably reduced when the enzyme was preincubated with the compound at room temperature. In a similar manner, the hypolipidemic agent clofibrate-free acid inhibited the growth of yeast by 50% at 3.0 mm. This inhibition was also prevented by acetate and not by pyruvate. In addition, clofibrate-free acid inhibited partially purified pyruvate dehydrogenase by 50% at a concentration of 37.0 mm.     

Evidence that phosphatidylcholine and phosphatidylethanolamine are synthesized by a single enzyme present in the endoplasmic reticulum of castor-bean endosperm.

Increasing concentrations of CDP-choline progressively inhibit the measured incorporation of CDP-[2-14C]ethanolamine into phosphatidylethanolamine catalysed by the ethanolaminephosphotransferase present in endoplasmic-reticulum membranes isolated from castor-bean endosperm cells. This inhibition parallels that observed during CDP-[Me-14C]choline incorporation and suggests that a single enzyme utilizes both these substrates.     

The Origin of Carbons of Dihydrofuran Ring and C-6 in the Biosynthesis of Rotenone

For the investigation of rotenone biosynthesis, acetate-2-¹⁴C, mevalonic acid-2-¹⁴C lactone and methionine-methyl-¹⁴C were administered to Derris elliptica plants, respectively, and the distribution of carbon-14 in the labeled rotenone was determined by degradation. When mevalonic acid-2-¹⁴C lactone was incorporated into rotenone, the radioactivity was found equally in the carbons at both C-7′ and C-8′, indicating that these carbons are derived from the carbon-2 of mevalonic lactone. In the case of methionine-methyl-¹⁴C about 80% of the total radioactivity was found to enter two methoxyl groups. This result demonstrates that methionine is an efficient precursor of the methoxyl group. Furthermore, it is also suggested that methionine may be a precursor of the carbon at C-6.